Pathogen Detection Using Metagenomic Next-Generation Sequencing of Plasma Samples from Patients with Sepsis in Uganda

Grundy, Brian S; Parikh, Hardik I.; Jacob, Shevin T.; Banura, Patrick; Moore, Chris C; Liu, Jie; Houpt, Eric R.

doi:10.1128/spectrum.04312-22

Cited by 8 publications

(5 citation statements)

References 40 publications

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“…Despite the existing selection bias, our study results indicate that patients with primary or secondary immunodeficiency particularly benefit from this type of diagnosis. Only few retrospective studies have so far evaluated mNGS diagnostics in patients with HIV infection [13]. To our knowledge, this is the first study to report on the use of mNGS for the detection of pathogens in HIV-infected patients in routine clinical practice.…”

Section: Discussionmentioning

confidence: 96%

Metagenomic next-generation sequencing as a diagnostic tool in the clinical routine of an infectious diseases department: a retrospective cohort study

Kalbitz,

Ermisch,

Kellner

et al. 2024

Infection

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Background Metagenomic next-generation sequencing (mNGS) of circulating cell-free DNA from plasma is a hypothesis-independent broadband diagnostic method for identification of potential pathogens. So far, it has only been investigated in special risk populations (e.g. patients with neutropenic fever). Purpose To investigate the extent to which mNGS (DISQVER® platform) can be used in routine clinical practice. Methods We collected whole blood specimens for mNGS testing, blood cultures (BC), and pathogen-specific PCR diagnostics. Clinical data and pathogen diagnostics were retrospectively reviewed by an infectious disease expert panel regarding the adjustment of anti-infective therapy. Results In 55 selected patients (median age 53 years, 67% male) with heterogeneous diagnoses, a total of 66 different microorganisms and viruses were detected using mNGS (51% viruses, 38% bacteria, 8% fungi, 3% parasites). The overall positivity rate of mNGS was 53% (29/55). Fifty-two out of 66 (79%) potential pathogens detected by mNGS were found in patients with primary or secondary immunodeficiency. The concordance rates of BC and pathogen-specific PCR diagnostics with mNGS testing were 14% (4/28) and 36% (10/28), respectively (p < 0.001). An additional bacterial pathogen (Streptococcus agalactiae) could only be detected by BC. Therapeutic consequences regarding anti-infective therapy were drawn from 23 pathogens (35% of detections), with 18 of these detections occurring in patients with immunodeficiency. Conclusions We conclude that mNGS is a useful diagnostic tool, but should only be performed selectively in addition to routine diagnostics of infectious diseases. The limited number of patients and the retrospective study design do not allow any further conclusions.

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Section: Discussionmentioning

confidence: 96%

Metagenomic next-generation sequencing as a diagnostic tool in the clinical routine of an infectious diseases department: a retrospective cohort study

Kalbitz,

Ermisch,

Kellner

et al. 2024

Infection

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“…Therefore, selecting the threshold criteria is crucial for reliable detection. Grundy et al (2023) 15 experimentally established that 2.4 reads per million (rPM) is the optimum threshold for viral pathogen detection in a metagenomics dataset. We, however used the more conservative filters NT rPM >= 10 and NT L (alignment length) >=35.…”

Section: Threshold For Viral Reads Detectionmentioning

confidence: 99%

Detection of Lumpy Skin Disease Virus Reads in the Human Upper Respiratory Tract Microbiome Requires Further Investigation

Tomar,

Khairnar

2024

Preprint

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Lumpy skin disease virus (LSDV), a double-stranded DNA virus from the Capripoxvirus genus, primarily affects Bos indicus, Bos taurus breeds, and water buffalo. Arthropod vectors, including mosquitoes and biting flies, are the main LSDV transmitters. Although LSDV is not zoonotic, this study unexpectedly detected LSDV reads in the upper respiratory tract microbiome of humans from rural and urban areas in Maharashtra, India. Nasopharyngeal and oropharyngeal swab samples collected for SARS-CoV-2 surveillance underwent whole-genome metagenomics sequencing, revealing LSDV reads in 25% of samples. SKA analysis provided insights into sample relatedness despite the low coverage of LSDV reads with the reference genome. Our findings, which include the detection of LSDV contigs aligning to specific locations on the reference genome, suggest a common source for LSDV reads, potentially shared water sources or milk/milk products. Further investigation is needed to ascertain whether these reads indicate actual LSDV infections, environmental uptake, or cross-reactivity with related viral sequences.

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“…This study exemplifies the efforts underway to refine and improve bioinformatic tools for metagenomics. Grundy also recently utilized metagenomic sequencing to detect pathogens in archived plasma samples from 254 patients with sepsis in Uganda [ 112 ]. Additionally, they conducted a study to compare the performance of qPCR and short-read Illumina sequencing for detecting HIV.…”

Section: Need For Identification Of the Best-use Practices That Accel...mentioning

confidence: 99%

Non-Targeted RNA Sequencing: Towards the Development of Universal Clinical Diagnosis Methods for Human and Veterinary Infectious Diseases

Spatz,

Afonso

2024

Veterinary Sciences

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Metagenomics offers the potential to replace and simplify classical methods used in the clinical diagnosis of human and veterinary infectious diseases. Metagenomics boasts a high pathogen discovery rate and high specificity, advantages absent in most classical approaches. However, its widespread adoption in clinical settings is still pending, with a slow transition from research to routine use. While longer turnaround times and higher costs were once concerns, these issues are currently being addressed by automation, better chemistries, improved sequencing platforms, better databases, and automated bioinformatics analysis. However, many technical options and steps, each producing highly variable outcomes, have reduced the technology’s operational value, discouraging its implementation in diagnostic labs. We present a case for utilizing non-targeted RNA sequencing (NT-RNA-seq) as an ideal metagenomics method for the detection of infectious disease-causing agents in humans and animals. Additionally, to create operational value, we propose to identify best practices for the “core” of steps that are invariably shared among many human and veterinary protocols. Reference materials, sequencing procedures, and bioinformatics standards should accelerate the validation processes necessary for the widespread adoption of this technology. Best practices could be determined through “implementation research” by a consortium of interested institutions working on common samples.

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Pathogen Detection Using Metagenomic Next-Generation Sequencing of Plasma Samples from Patients with Sepsis in Uganda

Abstract: The use of next-generation sequencing (NGS) in blood samples is an emerging technology for clinical microbiology labs. In this work, we performed NGS on plasma samples from a well-characterized cohort, where all samples had been previously tested by PCR for 43 pathogens.

Cited by 8 publications

References 40 publications

Metagenomic next-generation sequencing as a diagnostic tool in the clinical routine of an infectious diseases department: a retrospective cohort study

Metagenomic next-generation sequencing as a diagnostic tool in the clinical routine of an infectious diseases department: a retrospective cohort study

Detection of Lumpy Skin Disease Virus Reads in the Human Upper Respiratory Tract Microbiome Requires Further Investigation

Non-Targeted RNA Sequencing: Towards the Development of Universal Clinical Diagnosis Methods for Human and Veterinary Infectious Diseases

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